Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense

A abidopsis P o ein Phospha ase DBP1 Nuclea es a P o ein Ne wo k wi h a Role in Regula ing Plan De ense Jose ´Luis Ca asco 1. , Ma ı ´a Jose ´Cas ello ´ 1. , Kai Naumann 2 , Ines Lassowska 2 , Ma isa Na a e e-Go ´mez 1 , Die k Scheel 2 , Pablo Ve a 1,2 * 1Ins i u o de Biologı ´a Molecula y Celula de Plan as, Uni e sidad Poli e ´cnica de Valencia-C.S.I.C, Ciudad Poli e ´cnica de la Inno acio ´n, Valencia, Spain, 2Depa men o S ess and De elopmen al Biology, Leibniz Ins i u e o Plan Biochemis y, Halle, Ge many Abs ac A abidopsis haliana DBP1 belongs o he plan -speci ic amily o DNA-binding p o ein phospha ases. Al hough ecen ly iden i ied as a no el hos ac o media ing suscep ibili y o po y i us, li le is known abou DBP1 a ge s and pa ne s and he molecula mechanisms unde lying i s unc ion. Analyzing changes in he phosphop o eome o a loss-o - unc ion dbp1 mu an enabled he iden i ica ion o 14-3-3liso o m (GRF6), a p e iously epo ed DBP1 in e ac o , and MAP kinase (MAPK) MPK11 as componen s o a small p o ein ne wo k nuclea ed by DBP1, in which GRF6 s abili y is modula ed by MPK11 h ough phospho yla ion, while DBP1 in u n nega i ely egula es MPK11 ac i i y. In e es ingly, g 6 and mpk11 loss-o - unc ion mu an s showed al e ed esponse o in ec ion by he po y i us Plum pox i us (PPV), and he desc ibed molecula mechanism con olling GRF6 s abili y was ecapi ula ed upon PPV in ec ion. These esul s no only con ibu e o a be e knowledge o he biology o DBP ac o s, bu also o MAPK signalling in plan s, wi h he iden i ica ion o GRF6 as a likely MPK11 subs a e and o DBP1 as a p o ein phospha ase egula ing MPK11 ac i i y, and un eils he implica ion o his p o ein module in he esponse o PPV in ec ion in A abidopsis. Ci a ion: Ca asco JL, Cas ello ´MJ, Naumann K, Lassowska I, Na a e e-Go ´mez M, e al. (2014) A abidopsis P o ein Phospha ase DBP1 Nuclea es a P o ein Ne wo k wi h a Role in Regula ing Plan De ense. PLoS ONE 9(3): e90734. doi:10.1371/jou nal.pone.0090734 Edi o : Abidu Rahman, Iwa e Uni e si y, Japan Recei ed No embe 26, 2013; Accep ed Feb ua y 3, 2014; Published Ma ch 4, 2014 Copy igh : ß2014 Ca asco e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This wo k was suppo ed by he Spanish MICINN (G an s BFU2009-09771, EUI2009-04009 o PV), Gene ali a Valenciana (P ome eo2010/020 o PV) and he Ge man DFG (SCHE 235/15-1 o DS). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * E-mail: [email p o ec ed] .These au ho s con ibu ed equally o his wo k. In oduc ion DNA-binding p o ein phospha ases (DBPs) a e a unique amily o p o ein phospha ases o he 2C class ha a e dis inc i ely capable o binding DNA [1]. While p o ein phospha ase ac i i y lies in he C- e minal domain, he abili y o bind DNA esides in an N- e minal ex ension, which is ba ely conse ed beyond a mo i di ec ly in ol ed in binding [1,2]. DBP ac o s, al hough appa en ly unique o plan s, a e p esen h oughou he plan kingdom and seem o ha e unc ionally di e si ied du ing e olu ion [2]. Tobacco DBP1 (N DBP1), he ounding membe o his amily, was shown o be in ol ed in he ansc ip ional egula ion o a de ense- ela ed gene in he cou se o compa ible plan - i us in e ac ions [1]. Mo e ecen ly, in A abidopsis haliana, DBP1 was ound o media e disease suscep ibili y o po y i uses like Plum pox i us (PPV) and Tu nip mosaic i us (TuMV) [3]. Mo eo e , DIP2, a small pep ide o unknown unc ion ha unc ionally modula es DBP1 in A abidopsis, was iden i ied as an addi ional hos ac o in luencing PPV in ec ion, he eby emphasizing he signi ican implica ion o DBP1 in he in e ac ion [4]. DBP1 unc ion is also modula ed h ough he in e ac ion wi h 14-3-3 p o eins, highly conse ed ubiqui ous euka yo ic p o eins ecognized as impo an media o s in he egula ion o di e se biological p ocesses, in pa icula in signal ansduc ion and ansc ip ion [5]. Ca asco e al (2006) epo ed ha bo h in obacco and A abidopsis, DBP1 o hologs di ec ly in e ac wi h 14-3-3G and 14-3-3liso o ms, espec i ely, and his in e ac ion p o okes he nuclea expo o he complex o he cy oplasm [6]. Re en ion o DBP1 in he cy osol will in u n p e en binding o DBP1 o he p omo e egion o de ense- ela ed a ge genes, e en ually elie ing gene ep ession and concomi an ly p omo ing unc ion o DBP1 in he cy osol. Mi ogen-ac i a ed p o ein kinase (MAPK) cascades a e essen ial componen s o signal ansduc ion mechanisms e olu iona ily conse ed in all euka yo es. A ypical MAPK module consis s o h ee p o ein kinases, MAP kinase kinase kinase (MAP3K), MAP kinase kinase (MAPKK) and MAPK, which sequen ially phos- pho yla e and ac i a e each o he o media e cell esponses and de elopmen al pa hways [7]. Once ac i a ed, MAPK phospho - yla es and modula es speci ic a ge s, such as ansc ip ion ac o s. MAPK ac i a ion occu s h ough conse ed h eonine and y osine phospho yla ion by MAPKK, a dual speci ici y kinase [8]. Dephospho yla ion o ei he esidue by speci ic p o ein phospha ases inac i a es MAPK [9]. The p esen s udy p o ides u he insigh s in o he molecula mechanisms unde lying DBP1 unc ion by compa ing he phosphop o eome o loss-o - unc ion dbp1 mu an plan s o wild ype. Among he de ec ed p o eins, we iden i ied 14-3-3l(GRF6), an ea lie desc ibed DBP1 in e ac o , and MPK11, a MAPK PLOS ONE | www.plosone.o g 1 Ma ch 2014 | Volume 9 | Issue 3 | e90734 ac i a ed du ing he plan immune esponse [10]. Physical in e ac ions among hese p o eins we e demons a ed in i o by Bimolecula Fluo escence Complemen a ion analyses and coim- munop ecipi a ion. We showed ha DBP1 nega i ely egula ed MPK11 ac i i y, and ha MPK11 media ed phospho yla ion o GRF6, p omo ing ubiqui ina ion and inc eased GRF6 p o ein u n-o e . These esul s un eil a phospho yla ion-dependen , p o easome-media ed egula o y mechanism o GRF6, and iden i y a likely subs a e o MPK11, a MAPK wi h unknown unc ion, whose ac i i y is shown o be, in u n, modula ed by DBP1. In e es ingly, e e se gene ic s udies e ealed ha bo h GRF6 and MPK11 a e in ol ed in he esponse o A abidopsis plan s o in ec ion by he po y i us Plum pox i us, and he desc ibed egula ion o GRF6 p o ein s abili y by MPK11 was shown o occu du ing PPV in ec ion, appea ing as a no el signalling componen in he A abidopsis-PPV in e ac ion. Ma e ials and Me hods Plan Ma e ial and G ow h Condi ions A abidopsis plan s we e g own in a g ow h chambe unde a 10/14 h ligh /da k pho ope iod (23 o 19uC, 85% ela i e humidi y, 100 mEm 22 s 21 luo escen illumina ion). All mu an s we e in a Col-0 backg ound and we e ob ained om he Eu opean A abidopsis S ock Cen e a he Uni e si y o No hingham (h p://a abidopsis.in o/). dbp1 mu an was al eady desc ibed [3]. mpk11 and g 6 co espond o SALK_049352 and SALK_075219 lines, espec i ely (Salk Ins i u e Genomic Analysis Labo a o y, h p://signal.salk.edu/). Phosphop o ein En ichmen and Analysis A de ailed desc ip ion o he p o ocol used o phosphop o ein en ichmen is included in Suppo ing In o ma ion. T ansien Exp ession in Nico iana ben hamiana Lea es Tobacco plan s (Nico iana ben hamiana) we e g own in a phy ochambe unde sho day condi ions a 23uC/19uC. Almos ully expanded lea es we e in il a ed wi h a suspension o Ag obac e ium ume aciens C58 bea ing he ele an cons uc in 10 mM MES pH 5.6, 10 mM MgCl 2 , 150 mM ace osy ingone a an OD 600 = 0.5. A e 3 days, luo escence was analyzed in in il a ed lea es by con ocal mic oscopy. Fo co-in il a ion, Ag obac e ium cul u es g own sepa a ely and p ocessed as indica ed abo e, we e adjus ed o an O.D. = 0.5, and mixed p io o in il a ion. Ag obac e ium exp essing he i al silencing supp esso P19 was included in all in il a ions. Fluo escence Mic oscopy GFP/YFP luo escence in inocula ed plan s was moni o ed using Nikon SMZ800, and Leica MZ16F mic oscopes. Bimolecula Fluo escence Complemen a ion Analysis The ele an gene coding sequences we e ampli ied by PCR using speci ic oligonucleo ides bea ing a B si es, cloned in o pDONR207 (In i ogen), and inally ans e ed o pYFN43 and pYFC43 plasmids c ea ed by A. Fe ando (IBMCP, Valencia, Spain; h p://www.ibmcp.up .es), o gene a e C- e minal ans- la ional usions o he N- e minal (YFP N ) and C- e minal (YFP C ) agmen s o he yellow luo escen p o ein (YFP), espec i ely. Once ans e ed o Ag obac e ium, cons uc s we e ansien ly exp essed in N. ben hamiana lea es as desc ibed abo e. Fluo escence was de ec ed by con ocal mic oscopy h ee days ollowing in il a ion. Vi al Inocula ion PPV was inocula ed by gen ly making a single punc u e on he dis al pa o a lea wi h a s e ile oo hpick soaked in a suspension o A. ume aciens bea ing an in ec ious cDNA clone a an O.D. = 1 a 600 nm. The Ag obac e ium suspension was p epa ed as desc ibed abo e. RNA Ex ac ion, RT and qPCR To al RNA was ex ac ed using TRIzolH eagen (In i ogen) ollowing he manu ac u e ’s ecommenda ions and u he pu i ied by li hium chlo ide p ecipi a ion. Fo e e se ansc ip- ion, he Re e Aid TM H Minus Fi s S and cDNA Syn hesis Ki (Fe men as Li e Sciences) was used. Quan i a i e PCR (qPCR) ampli ica ions and measu emen s we e pe o med using an ABI PRISM 7000 sequence de ec ion sys em, and SYBR-G een (Pe kin-Elme Applied Biosys ems). ACTIN2/8 was chosen as he e e ence gene. The ollowing oligonucleo ides we e u ilized in he analyses: PPV-CP-FW: 59-GACTACGGCGTCAATGCTCAAC-39 PPV-CP-RV: 59- GTTTGCAGTTGAGGTCCTGACAC-39 ACTIN2/8-FW: 59-GGTAACATTGTGCTCAGTGGTGG- 39 ACTIN2/8-RV: 59-AACGACCTTATCTTCATGCTGC-39 MPK4-FW: 59-TTCCCAAACATGTCGGCTGGTG-39 MPK4-RV: 59-TGGCACAACGCCTCATCAACTG-39 MPK11-FW: 59-ACCCAAACAGACGCATTACAGTCG-39 MPK11-RV: 59-TCGTGTAGCGGTGCTAAGTAAGG-39 GRF6-FW: 59-TATGCAGGAGCAGATGGACGAG-39 GRF6-RV: 59-GGTGGCAGAAACATCGCGTAAC-39 Wes e n Blo and Immunop ecipi a ion P o ein c ude ex ac s we e p epa ed by homogenizing g ound ozen lea ma e ial wi h T is-bu e ed saline (TBS) supplemen ed wi h 5 mM DTT, p o ease inhibi o cock ail (Sigma-Ald ich), and p o ein phospha ase inhibi o s (PhosS op, Roche). P o ein con- cen a ion was measu ed using B ad o d eagen ; 25 mg o o al p o ein was sepa a ed by SDS-PAGE (12% ac ylamide w/ ) and ans e ed o ni ocellulose il e s. The il e was s ained wi h Ponceau-S a e ans e , and used as a loading con ol. Fo immunop ecipi a ions, ex ac s we e clea ed by wo successi e cen i uga ion s eps a 160006g and 4uC o 30 minu es, and hen incuba ed a 4uC o 2 h wi h conjuga ed an ibodies unde gen le ocking. Unbound p o eins we e emo ed by successi ely washing he esin wi h TBS, TBS 0.5% ( / ) T i on X-100 and TBS 0.3M NaCl, 1% ( / ) NP-40. We used monoclonal an i-HA clone HA-7 aga ose-conjuga e (Sigma- Ald ich), and abbi polyclonal GFP-sepha ose conjuga e (Abcam). Immunop ecipi a ed p o eins we e elu ed wi h Laemmli sample bu e by hea ing o 5 minu es a 95uC. Fo Wes e n blo s analysis he ollowing an ibodies we e used: mouse monoclonal an i-GFP an ibodies (Roche), abbi polyclonal an i-MaBP an ibodies (New England Biolabs), mouse monoclonal an i-phosphoMyBP clone P12 an ibodies (Millipo e), abbi polyclonal an i-HA an ibodies (Sigma), mouse monoclonal an i-phosphose ine Q5 an ibodies (Qiagen), and mouse monoclonal an i-ubiqui in Ub (P4D1) an ibodies (San a C uz Bio echnology). MAP Kinase Ac i i y Assay The assay was pe o med a e MPK11 immunop ecipi a ion, and p io o elu ion. Resin was esuspended wi h a eac ion mix consis ing o 50 mM T is-HCl pH 7.5, 10 mM MgCl 2 ,1mM DTT, 0.1 mM ATP and 0.4 mg/ml myelin basic p o ein (MyBP). Reac ions we e incuba ed o 30 min a 30uC in a shaking A abidopsis DBP1 Phospha ase PLOS ONE | www.plosone.o g 2 Ma ch 2014 | Volume 9 | Issue 3 | e90734 incuba o . Following incuba ion Laemmli sample bu e was added, and samples we e hea ed o 5 minu es a 95uC. Phospho yla ed MyBP was de ec ed by Wes e n blo using a speci ic mouse monoclonal an ibody as desc ibed abo e, ollowing manu ac u e ’s ins uc ions. Resul s dbp1 Mu an Phosphop o eome Analysis DBP1 is a p o ein phospha ase 2C o he DNA-binding DBP amily. Changes in he phosphop o eome o DBP1-de ec i e plan s could e eal di ec o indi ec a ge s o DBP1. Consequen ly, he phosphop o eome o wild- ype Col-0 and dbp1 mu an plan s was analysed applying an op imized wo k low o highly e icien phosphop o ein en ichmen om adul plan lea es, including ammonium sul a e p ecipi a ion and me al-oxide a ini y ch oma- og aphy [11] (see Me hods S1). Fo a sensi i e and de ailed sho gun analysis, LTQ O bi ap Velos echnology was used on h ee biological eplica es pe geno ype and ela i e quan i ica ion based on spec al coun was pe o med. E iciency and ep oduc- ibili y o his p ocedu e was analyzed by 1-D-SDS-PAGE and subsequen P oQ Diamond and CBB s aining. As lis ed in Table SI, 169 (phospho)p o eins showed di e en ial accumula ion in he dbp1 mu an ela i e o Col-0 ( - es , p,0.05). In e es ingly, among he iden i ied p o eins an ea lie desc ibed DBP1 in e ac o was ound. 14-3-3l(GRF6; A 5g10450) was p e iously shown o speci ically in e ac wi h DBP1 in i o [6]. In addi ion, iden i ica- ion o he MAP kinase pai MPK4 (A 4g01370)/MPK11 (A 1g01560) was also pa icula ly signi ican since i p o ides an insigh in o he signalling p ocesses DBP1 migh be pa o . Howe e , p o ein iden i ica ion in his case is ambiguous due o he high sequence simila i y be ween MPK4 and MPK11. Consis en wi h an 89% iden i y be ween hese wo p o eins, mos o he yp ic pep ides iden i ied we e p esen in bo h. The e o e, a his s age we could no asce ain he ac ual ela ionship o hese wo MAPKs wi h DBP1. MAPKs a e key componen s o signal ansduc ion pa hways in euka yo es. MPK4 is ac i a ed in esponse o pa hogen-associa ed molecula pa e ns (PAMPs) and has been epo ed o be in ol ed in plan immuni y as a nega i e egula o o he salicylic acid (SA)-media ed de ense pa hway [12,13]. MPK11 was also ound o be ac i a ed ollowing applica ion o he PAMP molecule lg22 [10], bu he signalling pa hways in which i ope a es emain unknown, as uniden i ied a e i s p o ein a ge s. As p e iously epo ed, he mpk4 mu an showed se e e dwa ism (Figu e S1B) [12,14]. The e o e, since DBP1 has no been ela ed so a o any de elopmen al al e a ion and he dbp1 mu an exhibi s wild- ype mo phology and a chi ec- u e, we ocused on he mpk11 mu an o u he s udies. Subcellula Localiza ion and Analysis o P o ein-p o ein In e ac ions The di e en ial p o ein accumula ion obse ed in he dbp1 mu an migh e eal a di ec o indi ec e ec o he absence o DBP1 p o ein phospha ase ac i i y. The e o e, we analysed whe he he selec ed p o eins in e ac wi h DBP1 in i o using Bimolecula Fluo escence Complemen a ion (BiFC). The ele an p o eins mus co-localize o in e ac , and localiza ion migh be al e ed as a consequence o he in e ac ion. The e o e, we i s examined he subcellula localiza ion o MPK11 and GRF6 when ansien ly exp essed as GFP usions in Nico iana ben hamiana lea es by ag oin il a ion as desc ibed [4]. Bo h obacco and A abidopsis DBP1 localize in he nucleus and he cy osol [6]. He e, he selec ed p o eins exhibi ed a simila subcellula dis ibu ion (Fig. 1). Nuclea localiza ion was e i ied using cen ome ic his one H3 used o Red Fluo escen P o ein (mRFP) as a nuclea ma ke [15]. The e o e, he espec i e localiza ion pa e ns enabled in e ac ion wi h DBP1. BiFC sugges ed ha he in e ac ions ac ually occu in i o a e ansien ly exp essing he ele an p o eins used o he N- e minal and C- e minal agmen s o he Yellow Fluo escen P o ein (YFP) in N. ben hamiana by ag oin il a- ion. The in e ac ion esul s in YFP luo opho e econs i u ion and a luo escence signal whe e he p o ein complex accumula es. Fig. 2A depic s con ocal mic oscopy images ob ained ollowing coexp ession o he indica ed usion p o eins. DBP1-GRF6 and DBP1-MPK11 complexes we e de ec ed bo h in he cy osol and in he nucleus. As abo e, mRFP- agged cen ome ic his one H3 was included in he analysis as indica i e o nuclea localiza ion. Fo GRF6, his was consis en wi h p e ious obse a ions ha obacco GRF6 and DBP1 homologues in e ac ed o media e nucleocy o- plasmic shu ling and DBP1 accumula ion in he cy osol [6]. In e ac ions we e alida ed by coimmunop ecipi a ion o usion p o eins ansien ly exp essed in lea es o N. ben hamiana (Fig. 2B and Figu e S2)). DBP1 used o mal ose binding p o ein (MaBP) was immunop ecipi a ed wi h an i-MaBP an ibodies. When coexp essed wi h MPK11 used o YFP, o GRF6 agged wi h he hemagglu inin (HA) epi ope, p esence o he pu a i e pa ne in he immunop ecipi a e was demons a ed by Wes e n blo using an i-GFP and an i-HA an ibodies, espec i ely. The e o e, bo h MPK11 and GRF6 in e ac in i o wi h DBP1. DBP1 Inac i a es MPK11 in i o Since MAPK ac i i y is egula ed by phospho yla ion and DBP1 is a p o ein phospha ase, DBP1 migh inac i a e MPK11. To es his hypo hesis, we ansien ly exp essed MPK11 in N. ben hamiana as a YFP N- e minal usion, alone o in combina ion wi h DBP1 used o MaBP. MPK11 was hen immunop ecipi a ed wi h an i-GFP an ibodies, and MAPK ac i i y was measu ed in he immunop ecipi a e using myelin basic p o ein (MyBP) as a subs a e and an i-phospho-MyBP an ibodies o p oduc de ec- ion. The expe imen was epea ed h ee imes. Albei he le el o ac i i y o MPK11 was a iable, likely because ac i a ion o exp essed MPK11 elies on endogenous kinases om N. ben hamiana, he de ec ed ac i i y was ma kedly educed in e e y case when DBP1 was coexp essed wi h MPK11. As shown in Fig. 3A o a ep esen a i e expe imen , simila MPK11 p o ein amoun s we e immunop ecipi a ed in bo h cases (uppe panel), bu when coexp essed wi h DBP1 (middle panel), MPK11 ac i i y was signi ican ly educed (lowe panel). The e o e, DBP1 is a p o ein phospha ase di ec ly egula ing MPK11 ac i i y. Since DBP1 inhibi ed MPK11, di e en ial (phospho)p o ein accumula ion in Col-0 and he dbp1 mu an migh no exclusi ely e lec di ec DBP1-media ed dephospho yla ion, bu also indi ec e ec s o inc eased MPK11 ac i i y in he mu an . Fo ha eason, and o help cla i y how his eme ging p o ein ne wo k ope a es, we analysed i GRF6 migh be a MPK11 subs a e. Using BiFC, we i s showed ha GRF6 in e ac ed wi h MPK11 in i o (Fig. 3B). The complex la gely accumula ed in he nucleus, indica ing ha he in e ac ion likely occu ed in he nuclea compa men . The in e ac ion was con i med by coimmunop ecipi a ion (Fig. 3C). When coexp essed by ag oin il a ion in N. ben hamiana oge he wi h GRF6-HA, YFP-MPK11 was de ec ed by Wes e n-blo in he an i-HA immunop ecipi a e. MPK11 Phospho yla es GRF6 in i o The MPK11-GRF6 in e ac ion sugges ed ha GRF6 migh be a MPK11 subs a e. To e i y his hypo hesis, GRF6 phospho - yla ion was analyzed by Wes e n blo a e immunop ecipi a ion, using wo di e en monoclonal an ibodies ecognizing phospho - A abidopsis DBP1 Phospha ase PLOS ONE | www.plosone.o g 3 Ma ch 2014 | Volume 9 | Issue 3 | e90734 yla ed se ine and h eonine esidues. The an i-phosphose ine and an i-phospho h eonine an ibodies a e comme cial an ibodies which ecognize he espec i e phospho yla ed esidues ega dless o su ounding amino acids. Di e ences we e only ound in se ine phospho yla ion. GRF6 was phospho yla ed a se ine esidues when exp essed in N. ben hamiana by an endogenous kinase ac i i y (Fig. 4A, le lane, bo om panel). Se ine phospho yla ion signi ican ly inc eased when GRF6was coexp essed wi h YFP- MPK11 (Fig. 4A, middle lane), sugges ing addi ional phospho - yla ion as media ed by MPK11. Whe he phospho yla ion may be occu ing di ec ly o indi ec ly ia ano he kinase ac i i y ha is modi ied by MPK11 emains s ill unknown. In he p esence o DBP1, he MPK11-dependen inc ease in GRF6 se ine phos- pho yla ion was p e en ed (Fig. 4A, igh lane), u he suppo ing MPK11 inac i a ion by DBP1. MPK11 P omo es Deg ada ion o GRF6 in i o When ansien ly coexp essed wi h MPK11 in N. ben hamiana, GRF6 ep oducibly accumula ed a lowe le els han when exp essed alone (Fig. 4B). This sugges ed lowe GRF6 s abili y in he p esence o MPK11. The ubiqui in-p o easome sys em (UPS) is a majo componen o he cell machine y engaged in he con ol o p o ein abundance, and plays an impo an ole in he egula ion o mul iple biological p ocesses [16]. UPS a ge p o eins a e ubiqui ina ed by E3 ubiqui in ligases, ecognized by he 26S p o easome complex and deg aded. The e o e, we analyzed GRF6 ubiqui ina ion ollowing ansien exp ession in N. ben hamiana and immunop ecipi a ion wi h an i-HA an ibodies. Wes e n blo analysis using an i-ubiqui in monoclonal an ibodies e ealed ha a ac ion o immunop ecipi a ed GRF6 was ubiqui ina ed (Fig. 4C, le panel), accumula ing p edominan ly as a likely e aubiqui ina ed o m acco ding o i s es ima ed molecula mass. To con i m he iden i y o his ubiqui ina ed p o ein as GRF6, a Wes e n blo was immunodeco a ed wi h an i- HA an ibodies, e ealing de ec ion o he same p o ein along wi h he as mig a ing and mo e abundan unmodi ied GRF6 p o ein (Fig. 4C, igh panel). In e es ingly, abundance o he ubiqui i- na ed ac ion inc eased when GRF6 was coexp essed wi h MPK11, sugges ing a highe ubiqui ina ion a e upon MPK11- media ed GRF6 phospho yla ion. This co ela ed wi h he inc eased GRF6 ins abili y we obse ed in he p esence o MPK11, as shown in Fig. 4B. Rein o cing he unc ional in e ela ionship exis ing among hese h ee p o eins, DBP1 p e en ed ubiqui ina ion o GRF6. When DBP1 and MPK11 we e simul aneously exp essed along wi h GRF6 in N. ben hamiana lea es, he p esence o DBP1 es ic ed MPK11-p omo ed GRF6 ubiqui ina ion (Fig. 4C). This was likely due o he inhibi o y e ec o DBP1 on MPK11 ac i i y which, as shown abo e, also p e en ed GRF6 phospho yla ion media ed by MPK11 (Fig. 4A). Candida e DBP1 Ta ge s had Di e en Roles in PPV In ec ion DBP1 unc ions as a hos suscep ibili y ac o du ing PPV in ec ion [3], whe eas DIP2, a small polypep ide unc ionally modula ing DBP1, con ibu es o esis ance [4]. The implica ion o he selec ed p o eins in he A abidopsis-PPV in e ac ion was examined by selec ing homozygous mu an plan s bea ing a T- DNA inse ion in he ele an gene ha ma kedly educes gene exp ession (Fig. S1A). Plan s o hese lines we e inocula ed wi h a PPV-GFP i al s ain, and in ec ion p og ession was moni o ed by luo escence mic oscopy and by RT-qPCR using speci ic p ime s o he i al coa p o ein gene. In e es ingly, mpk11 showed inc eased suscep ibili y o PPV, whe eas g 6 exhibi ed enhanced esis ance (Fig. 5A, B). Complemen a ion o he analyzed mu an s wi h genomic agmen s bea ing he wild- ype gene sequence showed ha he obse ed pheno ypes we e indeed due o loss o unc ion o he ele an genes (Figu e S1C). As bo h genes appea ed o be implica ed in he A abidopsis- PPV in e ac ion, we analyzed hei exp ession dynamics ela i e o i al accumula ion in Col-0. PPV-inocula ed plan s we e inspec ed unde he luo escence mic oscope and in ec ed lea es we e g ouped in o ou di e en ca ego ies based on he deg ee o i al coloniza ion. Lea ma e ial exhibi ing no (1), low (2), medium (3), Figu e 1. Subcellula dis ibu ion o he iden i ied DBP1 pu a i e a ge s. Full-leng h coding sequences we e used o he N- e minus o g een luo escen p o ein (GFP) o o he C- e minus o yellow luo escen p o ein (YFP), and ansien ly exp essed in Nico iana ben hamiana lea es by ag oin il a ion. Lea sec ions we e inspec ed by con ocal mic oscopy 3 d a e in il a ion. Fusion p o eins we e exp essed in epide mal cells.A la ge cen al acuole occupies mos o he cellula space, cons aining cy osol o a na ow pe iphe al a ea. Co-exp ession wi h mRFP- agged cen ome ic his one H3 e eals nuclei localiza ion. doi:10.1371/jou nal.pone.0090734.g001 A abidopsis DBP1 Phospha ase PLOS ONE | www.plosone.o g 4 Ma ch 2014 | Volume 9 | Issue 3 | e90734 and high (4) i al accumula ion we e ha es ed, and gene exp ession was analyzed by RT-qPCR, e e ed o ACT2/8 and no malized using simila samples om unin ec ed plan s (Fig. 5C). MPK11 ansc ip accumula ion inc eased du ing in ec ion, while GRF6 exp ession was ep essed ollowing MPK11 induc ion. Fig. 5D shows inc easing i al accumula ion h ough he selec ed ca ego ies as quan i ied by RT-qPCR. To demons a e he biological ele ance o hese indings, we analysed GRF6 accumula ion du ing he cou se o PPV in ec ion in N. ben hamiana. P o ein le el was measu ed a e 3 d o exp ession a di e en ime poin s o in ec ion (Fig. 6A, B). As desc ibed abo e, GRF6 accumula ion was ep oducibly dimin- ished when coexp essed wi h MPK11. He e, he magni ude o he dec ease obse ed in non-inocula ed issue (0 dpi) is lowe han ha shown ea lie . This is no su p ising, since de-s abiliza ion o GRF6 media ed by MPK11 depends on he exp ession le el o MPK11 and also on he deg ee o ac i a ion o he kinase, which elies on endogenous MAPKKs. A signi ican u he educ ion in GRF6 p o ein accumula ion was obse ed 4 d pos -inocula ion wi h PPV. This educ ion was speci ic and no obse ed o cy osolic asco ba e pe oxidase (APX), he e used as a nega i e con ol, nei he by Ponceau-S s aining o he p o ein ex ac s. GRF6 p o ein decline was ma kedly accele a ed in he p esence o Figu e 2. Iden i ied p o eins in e ac in i o wi h DBP1. A, BiFC assay wi h YFP usions ansien ly exp essed in N. ben hamiana lea es by ag oin il a ion. Bo h ecip ocal combina ions a e shown o each in e ac ion. As a nega i e con ol, DBP1 and he un ela ed ansc ip ion ac o BREVIS RADIX (BRX) YFP usions we e used. Cen ome ic his one H3 used o mRFP was co-exp essed as a ma ke o nuclea localiza ion. B, Coimmunop ecipi a ion alida ed he epo ed in e ac ions. Rele an usion p o eins we e ansien ly exp essed in N. ben hamiana lea es by ag oin il a ion as indica ed, and immunop ecipi a ed wi h an i-MaBP an ibodies. Wes e n blo s o he immunop ecipi a ed ac ions deco- a ed wi h he indica ed an ibodies a e shown. An i-GFP an ibodies allow o de ec ion o YFP. doi:10.1371/jou nal.pone.0090734.g002 Figu e 3. MPK11 in e ac s wi h GRF6 and is nega i ely modula ed by DBP1. A, DBP1 inhibi ed MPK11 kinase ac i i y. MPK11 and DBP1 we e ansien ly exp essed as ansla ional usions o YFP and MaBP, espec i ely, in N. ben hamiana lea es by ag oin il a- ion. YFP-MPK11 was immunop ecipi a ed 3 d a e in il a ion wi h an i-GFP an ibodies and MAPK ac i i y was measu ed in he immuno- p ecipi a e by Wes e n blo wi h an ibodies speci ically ecognizing phospho yla ed MyBP (lowe panel). A ac ion o he immunop ecip- i a ed p o ein was analyzed using an i-GFP (uppe panel) and an i-MaBP (middle panel) an ibodies. B, MPK11 in e ac ed wi h GRF6 in i o. BiFC analysis in N. ben hamiana lea es showing speci ic MPK11-GRF6 in e ac ion. The MPK11-GRF6 complex was de ec ed p edominan ly in he nucleus. C, GRF6 and MPK11 co-immunop ecipi a ion. GRF6-HA was exp essed in N. ben hamiana lea es and immunop ecipi a ed wi h an i- HA an ibodies. YFP-MPK11 was de ec ed by Wes e n blo wi h an i-GFP an ibodies in he an i-HA immunop ecipi a e when coexp essed wi h GRF6 (uppe panel), con i ming in e ac ion be ween he wo p o eins. Lowe panel shows GRF6-HA immunode ec ion in he immunop ecip- i a e by Wes e n blo . doi:10.1371/jou nal.pone.0090734.g003 A abidopsis DBP1 Phospha ase PLOS ONE | www.plosone.o g 5 Ma ch 2014 | Volume 9 | Issue 3 | e90734 MPK11, co obo a ing he inc eased GRF6 u n-o e caused by MPK11. To asce ain he implica ion o he p o easome in his p ocess, GRF6 and MPK11 we e ansien ly exp essed in lea es o N. ben hamiana which had been p e iously inocula ed wi h PPV in o de o p omo e ac i a ion o MPK11 and GRF6 deg ada ion. As shown in Fig. 6C, ea men wi h he p o easome inhibi o MG-132 24 h p io o collec ing he issue e e ed he educ ion in GRF6 s abili y obse ed when coexp essed wi h MPK11, indica ing ha he p o easome is media ing GRF6 deg ada ion p omo ed by MPK11. In con as , in he absence o MPK11, addi ion o MG-132 did no ha e any signi ican e ec on GRF6 p o ein accumula ion, as expec ed i phospho yla ion o GRF6 by MPK11 is he molecula e en ha a ge s GRF6 o deg ada ion ia he p o easome. Discussion DBPs a e plan -speci ic DNA-binding p o ein phospha ases [1,2], and ha e been ound o play a ole in plan - i us in e ac ions [1,3,4]. Howe e , li le is known abou he molecula mechanisms in which DBP ac o s a e in ol ed. The e o e, we sea ched o pu a i e DBP1 a ge s by analysing phosphop o eome changes esul ing om DBP1 loss-o - unc ion. F om he iden i ied p o eins we selec ed 14-3-3l(GRF6) and MPK11 o u he analyses. GRF6 is liso o m wi hin he 14-3-3 amily, highly conse ed p o eins in ol ed in egula ing mul iple biological p ocesses and signalling pa hways in euka yo es. 14-3-3 p o eins usually bind phosphopep ide mo i s and modi y unc ion o hei a ge s a di e en le els [17]. GRF6 was p e iously shown o in e ac wi h DBP1 using he yeas wo-hyb id sys em [6], an in e ac ion conse ed in obacco and A abidopsis. MPK11 is one o he abou 20 MAPKs p esen in A abidopsis. Toge he wi h MAPKKs and MAP3Ks, MAPKs o m signalling cascades media ing de elop- men al p ocesses and esponses o bo h bio ic and abio ic s esses in all euka yo ic o ganisms. Iden i ica ion o subs a es and ac o s egula ing kinase ac i i y is essen ial o a be e unde s anding o Figu e 4. MPK11 phospho yla ed GRF6 p omo ing ubiqui ina ion and inc eased u no e . A, De ec ion o GRF6 phospho yla ion. GRF6- HA was ansien ly exp essed, alone o oge he wi h YFP-MPK11 and MaBP-DBP1 as indica ed in N. ben hamiana lea es by ag oin il a ion, and immunop ecipi a ed using an i-HA an ibodies. The h ee uppe panels show de ec ion by Wes e n blo o he di e en exp essed p o eins wi h he indica ed an ibodies. The wo lowe panels show immunop ecipi a ion o GRF6-HA using an i-HA an ibodies: an aliquo o he immunop ecipi a ed ac ion was loaded abo e as a loading con ol, whe eas in he bo om panel se ine phospho yla ion de ec ed using an i-phosphose ine monoclonal an ibody is shown. B, MPK11 media ed inc eased GRF6 u no e . GRF6-HA and YFP-MPK11 we e exp essed in N. ben hamiana lea es by ag oin il a ion. Wes e n blo o p o ein ex ac s om in il a ed lea es 3 d pos -in il a ion immunodeco a ed wi h he indica ed an ibodies o speci ic exp essed p o ein de ec ion. Ponceau-S s aining o he ni ocellulose memb ane is shown a he bo om. C, GRF6 was ubiqui ina ed in i o and MPK11 inc eased ubiqui ina ion a e. The indica ed p o eins we e ansien ly exp essed in N. ben hamiana lea es by ag oin il a ion, and GRF6- HA was immunop ecipi a ed wi h an i-HA an ibodies 3 d pos -in il a ion. Le panel, Wes e n blo o he an i-HA immunop ecipi a e immunodeco a ed wi h an i-ubiqui in an ibodies. Righ panel, he same blo shown in he le panel was s ipped, and immunodeco a ed wi h an i-HA an ibodies, indica ing ha he de ec ed ubiqui ina ed p o ein co esponds o GRF6-HA and e ealing unmodi ied GRF6. doi:10.1371/jou nal.pone.0090734.g004 A abidopsis DBP1 Phospha ase PLOS ONE | www.plosone.o g 6 Ma ch 2014 | Volume 9 | Issue 3 | e90734 cell esponses o de elopmen al, ho mone and en i onmen al signals. MAPKs a e ac i a ed by phospho yla ion, whe eas dephospho yla ion leads o inac i a ion. In yeas and mammals, y osine and dual speci ici y phospha ases a e conside ed he majo MAPK phospha ases [18]. In plan s, se ine/ h eonine p o ein phospha ases 2C ha e also been shown o dephospho yla e and modula e MAPK ac i i y [19–23]. When ac i a ed, MAPKs phospho yla e a a ie y o subs a es, he eby modula ing hei unc ion. He e we show ha DBP1 nega i ely egula ed MPK11 ac i i y and ha GRF6 seems o be a MPK11 subs a e. In A abidopsis, only a ew MAPK a ge s ha e been iden i ied in i o so a . Popescu e al. (2009) unde ook an ex ensi e sea ch o subs a es o 10 MAPKs using p o ein mic oa ays, bu MPK11 was no included in he s udy [24]. We demons a ed ha GRF6 in e ac s wi h MPK11, and ha MPK11 p omo es phospho yla- ion o GRF6 in i o. Mo eo e , DBP1 p e en ed MPK11- media ed GRF6 phospho yla ion. In e es ingly, GRF6 accumu- la ed a lowe le els when coexp essed wi h MPK11, sugges ing a phospho yla ion-dependen egula ion o GRF6 p o ein abun- dance. Indeed, GRF6 was ubiqui ina ed in i o, and ubiqui ina ion was enhanced in he p esence o MPK11, leading o highe deg ada ion a es. In con as , he p esence o DBP1 p o ec ed GRF6 om he inc eased ubiqui ina ion p omo ed by MPK11. This is likely a consequence o he inhibi ion o MPK11 ac i i y exe ed by DBP1. Mo eo e , GRF6 deg ada ion was a enua ed in he p esence o he p o easome inhibi o MG-132, indica ing ha GRF6 is deg aded ia he p o easome. These esul s p o ide compelling e idence o a egula o y p o ein ne wo k nuclea ed by DBP1 which modula es s abili y o GRF6, wi h GRF6 in u n media ing nucleo-cy oplasmic shu ling o DBP1. A ubiqui in ligase was ecen ly epo ed o a ge 14-3-3 p o eins in a s imulus- speci ic manne in he cellula C/N balance esponse [25]. The e o e, o he 14-3-3 p o eins a e subjec ed o a simila egula ion h ough induced deg ada ion ia UPS. Figu e 5. Pu a i e DBP1 a ge s played a ole in PPV in ec ion, posi i ely o nega i ely a ec ing PPV mul iplica ion. A, P og ession o PPV in ec ion in loss-o - unc ion mu an s. Plan s o he indica ed geno ypes we e inocula ed wi h PPV-GFP and in ec ion ex en was analyzed by luo escence mic oscopy o moni o GFP dis ibu ion. B, PPV accumula ion in loss-o - unc ion mu an s. Vi al accumula ion was measu ed by RT-qPCR using p ime s speci ically ampli ying he i al coa p o ein gene. Da a we e no malized using ACT2/8 as a e e ence gene. Rela i e accumula ion wi h espec o Col-0 is shown as he mean o h ee independen expe imen s 6SD. C, Gene exp ession analysis du ing he cou se o PPV-GFP in ec ion in Col-0 plan s. Inocula ed lea issue showing p oduc i e in ec ion was pooled in o ou di e en ca ego ies acco ding o he deg ee o i al coloniza ion de e mined unde he luo escence mic oscope. Lea ma e ial wi h no isible (1), low (2), medium (3), and high (4) i al accumula ion was ha es ed, and gene exp ession was analyzed by RT-qPCR using ACT2/8 as a e e ence gene. Exp ession da a we e no malized o hose ob ained o simila samples collec ed om unin ec ed plan s. The mean o h ee independen expe imen s 6SD is shown. D, PPV accumula ion in he selec ed in ec ion ca ego ies. Vi al accumula ion was quan i ied by RT-qPCR as abo e, and e e ed o he alue in (1). Al hough no isible signal was obse ed in hese lea es when assessed mic oscopically, he e was an incipien in ec ion de ec able by RT-qPCR. doi:10.1371/jou nal.pone.0090734.g005 A abidopsis DBP1 Phospha ase PLOS ONE | www.plosone.o g 7 Ma ch 2014 | Volume 9 | Issue 3 | e90734 MPK11 p omo ed enhanced GRF6 deg ada ion, nega i ely egula ing GRF6 unc ion. BiFC analysis p o ides a cellula basis o be e in e p e hese biochemical da a. As DBP1, GRF6 also localized bo h o he nucleus and he cy osol (Fig. 1), and, in u n, in e ac ion be ween hese wo ac o s, as de ec ed by BiFC, was also obse ed in he wo cellula compa men s (Fig. 2A). Accumula ion o GRF6-DBP1 complex in he cy osol is cong uen wi h DBP1 elocaliza ion om he nucleus o he cy osol, as p omo ed by 14-3-3 G in obacco [6]. In con as , he in e ac ion be ween GRF6 and MPK11 was de ec ed p edominan ly in he nucleus. MPK11 could be p e en ing GRF6-DBP1 in e ac ion in he nucleus by p omo ing GRF6 deg ada ion. Phospho yla ion is well known o in luence ubiqui ina ion ei he posi i ely o nega i ely [26]. Thus, he ac i i y o E3 ubiqui in ligases may be egula ed by phospho yla ion. Al e na i ely, phospho yla ion o he a ge p o ein can enable o p e en E3 ubiqui in ligase ecogni ion, o egula e E3 ubiqui in ligase access o i s a ge by modi ying i s subcellula localiza ion. In he case o GRF6, bo h mechanisms a e possible. Phospho yla ion by MPK11 migh c ea e a binding si e o E3 ubiqui in ligase, o could ec ui GRF6 o he nucleus, whe e he E3 ubiqui in ligase migh be loca ed. Al hough he MPK11-GRF6 complex accumula es in he nucleus, we canno exclude ha he in e ac ion akes place in he cy osol, and p omo es apid nuclea a ge ing o GRF6 o ubiqui ina ion, he eby p e en ing in e ac ion wi h DBP1. Li le is known abou he biology o DBP ac o s. DBP1 was ecen ly epo ed o ac as a po y i us suscep ibili y ac o , since he absence o DBP1 unc ion hinde ed in ec ion by he po y i uses Plum pox i us (PPV) and Tu nip mosaic i us (TuMV) [3]. Complex se s o hos -pa hogen in e ac ions de e mine he ou come o plan i us in ec ion. These in e ac ions, he unde - lying mechanisms and he in ol ed signalling pa hways a e la gely unknown. We showed ha bo h MPK11 and GRF6 also play a ole in he A abidopsis-PPV in e ac ion, since he espec i e loss-o - unc ion mu an s exhibi ed an al e ed esponse o PPV in ec ion. MPK11 eme ges as a signalling ac o likely media ing he de ense esponse o PPV in ec ion, since loss o unc ion leads o inc eased suscep ibili y. Th ee weeks a e inocula ion, PPV accumula ion Figu e 6. MPK11 p omo es inc eased GRF6 p o ein u n-o e du ing PPV in ec ion. A, Analysis o GRF6 accumula ion in in ec ion by PPV. GRF6-HA was ansien ly exp essed in N. ben hamiana lea es alone o oge he wi h MPK11 as indica ed. P o ein accumula ion was analyzed 3 days pos -in il a ion in lea es ha we e no inocula ed wi h PPV (0 dpi), lea es ha we e inocula ed wi h PPV one day a e GRF6-HA ag oin il a ion (2 dpi; in hese lea es, PPV in ec ion is a day 2 when GRF6 p o ein accumula ion was analyzed), and lea es ha we e inocula ed wi h PPV one day be o e GRF6-HA ag oin il a ion (4 dpi; in hese lea es, PPV in ec ion is a day 4 when GRF6-HA accumula ion was eco ded). As an in e nal con ol, accumula ion o asco ba e pe oxidase (APX) is shown. Ponceau-S s aining o memb ane indica es equal p o ein loading. PPV and ACTIN2/8 RNA accumula ion was de e mined by RT-PCR. B, Quan i ica ion o GRF6 and APX bands in Wes e n blo using ImageJ so wa e. Values we e e e ed o hose in he absence o PPV and MPK11. The expe imen was epea ed h ee imes wi h simila esul s. C, P o easome media es GRF6 deg ada ion p omo ed by MPK11. GRF6-HA and YFP-MPK we e ansien ly exp essed in N. ben hamiana lea es 4 days a e inocula ion o he same lea es wi h PPV. Two days la e , lea es we e in il a ed ei he wi h he p o easome inhibi o MG-132 o wi h he same concen a ion o he sol en DMSO, and a e 24 h, lea ma e ial was collec ed. P o ein accumula ion was analyzed by Wes e n blo using speci ic an ibodies as indica ed. doi:10.1371/jou nal.pone.0090734.g006 A abidopsis DBP1 Phospha ase PLOS ONE | www.plosone.o g 8 Ma ch 2014 | Volume 9 | Issue 3 | e90734 was app oxima ely 10- old highe in he mpk11 mu an han in Col-0 plan s, as de e mined by RT-qPCR (Fig. 5A). The ac ha MPK11 exp ession was mode a ely and ansien ly induced du ing in ec ion suppo s his hypo hesis (Fig. 5C). This assigns a biological ole o MPK11, and sheds ligh on he signalling p ocesses igge ed in esponse o PPV in ec ion. The e o e, MPK11 shows p omise o enginee ing imp o ed esis ance agains PPV. In con as , GRF6-de icien plan s displayed enhanced esis ance o PPV in ec ion, a pheno ype simila o ha o dbp1 mu an s, sugges ing ha DBP1 and GRF6 likely coope a e o con e suscep ibili y, whe eas MPK11 would be an agonizing his e ec . Since MPK11 p omo es GRF6 deg ada ion, o es whe he his egula ion plays a ole in he A abidopsis-PPV in e ac ion, we analyzed GRF6 accumula ion du ing he cou se o PPV in ec ion in N. ben hamiana lea es (Fig. 6A). In PPV-inocula ed lea es, GRF6 p o ein le el was signi ican ly educed as he i us accumula ed in he locally in ec ed issue. Howe e , when co- exp essed wi h MPK11, GRF6 u no e was speci ically accele - a ed. This u he sugges s ha MPK11 would be in ol ed in a plan local de ense esponse aimed a educing GRF6 p o ein le el in ea ly s ages o PPV in ec ion inc easing i s p o easome- media ed deg ada ion. The e o e, ou esul s ha e a signi ican impac no only in he biology o DBP ac o s, bu also con ibu e o a be e knowledge o MAPK signalling pa hways in A abidopsis h ough he iden i ica ion o GRF6 as a likely MPK11 subs a e and o DBP1 as a p o ein phospha ase egula ing MPK11 ac i i y, and un eils he implica ion o his p o ein module in he esponse o PPV in ec ion in A abidopsis. Suppo ing In o ma ion Figu e S1 T-DNA inse ion mu an s used in his s udy. A, RT-qPCR analysis o gene exp ession. Da a we e no malized using ACT2/8 as a e e ence gene and a e exp essed ela i e o he espec i e exp ession le el in wild- ype Col-0 plan s. B, Images o 4 week-old ep esen a i e plan s o each mu an line compa ed o he wild- ype Col-0 eco ype. No isible al e a ion was obse ed in plan mo phology and a chi ec u e a any de elopmen al s age, excep o mpk4 mu an which showed se e e de elopmen al de ec s. C, mpk11 and g 6 mu an lines we e ans o med wi h genomic agmen s encompassing he co esponding wild- ype s uc u al genes and mo e han 1500 bp o p omo e sequence. Homozygous plan s bea ing single inse ions we e inocula ed wi h PPV and p og ession o in ec ion was analyzed by RT-qPCR using p ime s o he i al coa p o ein gene. Exp ession alues we e no malized using ACT2/8 and e e ed o Col-0. (TIF) Figu e S2 Speci ici y o he an ibodies used in immu- nop ecipi a ion assays. The indica ed p o ein combina ions we e ansien ly exp essed in N. ben hamiana lea es by ag oin il a- ion, and immunop ecipi a ed wi h he an ibodies indica ed on op. The co esponding immunop ecipi a ed ac ions we e analyzed by Wes e n blo using he an ibodies e e ed on he le . (TIF) Me hods S1 (DOCX) Table S1 (XLS) Acknowledgmen s Au ho s wish o hank V. Rami ez, J. Ga cı ´a-And ade and A. Lo´pez om P. Ve a’s lab o help ul discussions. Au ho Con ibu ions Concei ed and designed he expe imen s: JLC MJC KN IL MNG. Pe o med he expe imen s: JLC MJC KN IL MNG. Analyzed he da a: JLC MJC KN IL MNG DS PV. W o e he pape : JLC PV. Re e ences 1. Ca asco JL, Ancillo G, Mayda E, Ve a P (2003) A no el ansc ip ion ac o in ol ed in plan de ense endowed wi h p o ein phospha ase ac i i y. EMBO J 22: 3376–3384. 2. Ca asco JL, Ancillo G, Cas ello´ MJ, Ve a P (2005) A no el DNA-binding mo i , hallma k o a new amily o plan ansc ip ion ac o s. Plan Physiol 137: 602– 606. 3. Cas ello´ MJ, Ca asco JL, Ve a P (2010) DNA-binding p o ein phospha ase A DBP1 Media es Suscep ibili y o wo Po y i uses in A abidopsis. Plan Physiol 153: 1521–1525. 4. Cas ello´ MJ, Ca asco JL, Na a e e-Go´mez M, Daniel J, G ano D e al. (2011) A plan small polypep ide is a no el componen o DNA-binding p o ein phospha ase 1-media ed esis ance o Plum pox i us in A abidopsis. Plan Physiol 157: 2206–2215. 5. Denison FC, Paul AL, Zupanska AK, Fe l RJ (2011) 14-3-3 p o eins in plan physiology. Sem Cell De Biol 22: 720–727. 6. 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